The cochlea is composed of a variety of cell types including sensory, supporting and neural elements. Taken together, these cells comprise a functionally intricate and cohesive electrical unit that initiates the analysis of acoustic information within our environment. We capitalize on the in vitro approach, including isolated cochlea explants, single cell, and more recently stable cell lines to elucidate cochlear cell function; the strategy is to characterize basic properties prior to integration into a cohesive understanding of the organ. Currently, the overarching aim of this project is focused on determining the biophysical properties of key membrane components of the outer hair cell (OHC), one of the major players in auditory function, using a balance of electrophysiology, molecular biology, modelling and high resolution cryo-EM. Though we have made significant progress on many fronts since our last renewal in 2010, we now focus on three specific research topics that evolve from our most significant accomplishments. In particular, one of the main areas of our investigations has been and will be the influence of anions on the OHC molecular motor?s (prestin, SLC26a5) electro-mechanical activity. This ion is at the root of cochlear amplification (Santos-Sacchi et al., 2006). Indeed, the NIDCD?s 2012-2016 Strategic Plan specifically identifies understanding anion control of hearing as a key goal. The three aims are 1) to evaluate the chloride-dependent kinetic behavior of OHC nonlinear capacitance and electromotility, 2) to characterize intracellular chloride movements in the prestin-transfected HEK cell and OHC with a new prestin-fused YFP chloride sensor we created, and 3) to determine the high resolution structure of prestin (SLC26a5) and its closest mammalian family member SLC26a6 using cryo-EM, with the goal of identifying conformational changes due to chloride and voltage. We hypothesize that understanding these molecular activities will aid in understanding how the OHC enables us to hear so well and in turn how micro-environmental influences may lead to pathologies of the OHC that afflict millions.